Abstract
Transient transformation is a notably efficient and expeditious method for obtaining foreign gene expression products, making it a prominent avenue for investigating functional genes in pasture. The present study aimed to explore the non-in vitro transient transformation technique of Lotus japonicus, a leguminous herbage, to analyze functional genes in the future. This was achieved by evaluating the expression products of GUS reporter gene and the bioactivity of functional gene. The results indicated that the transient expression system was successfully optimized as follows: the utilization of parameters, including an OD600 absorption of 0.8 for the bacterium, a vacuum pressure of 12 Kpa, and an infiltration time of 15 min, yielded a transient transformation rate of 57.78%. The GUS recombinant protein demonstrated optimal accumulation on the 8th day, while the functional evaluation and utilization of the exogenous gene were observed within a time frame of 2–12 days. The entire branch-leaf explant demonstrated an efficient expression of the GUS recombinant protein within a 12-day timeframe while maintaining viability. This finding suggests that this system not only facilitates the extended analysis of gene function but also accurately reflects the regulatory capabilities of the entire living plant. Furthermore, the results confirmed the viability of the transient transformation system by demonstrating improved tolerance to high temperature, drought, and salt stress in Lotus japonicus expressing foreign LjHsp70-1 gene. The successful establishment of this transient expression system offers a valuable way and framework for investigating the functional characteristics of vital genes in Lotus japonicus or other microphyllous plants.
Key Message
In this high-efficiency gene transient expression system for Lotus japonicus, the duration of exogenous gene expression reaches 12 days by using the branch-leaf explant for genetic transformation.
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Data availability
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Abbreviations
- CAT:
-
Catalase
- EV:
-
Empty vector
- GUS:
-
β-Glucuronidase
- POD:
-
Peroxidase
- qRT-PCR:
-
Quantitative real-time polymerase chain reaction
- RWC:
-
Relative water content
- SOD:
-
Superoxide dismutase
- TP:
-
Transgenic plant
- WT:
-
Wild type
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Acknowledgements
The authors thank master student Zouxian Gong (Clinical Medical College of Guizhou Medical University, Guiyang 550004, Guizhou Province, China.) for providing writing assistance.
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This research was funded by the National Natural Science Foundation of China (No.32260338) and (No.31660685), and the Guizhou Province Science and Technology Project (2023ZK119).
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LS and FY conceived and designed the experiments. XY performed the experiments. XY carried out the bioinformatics analysis. XY wrote the manuscript. XL and YW gave insightful suggestions. LS improved the manuscript errors and English language. All authors read and approved the manuscript.
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Yin, X., Luo, X., Yang, F. et al. An effective transient expression system for gene function identification in Lotus japonicus. Plant Cell Tiss Organ Cult 156, 57 (2024). https://doi.org/10.1007/s11240-024-02688-7
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DOI: https://doi.org/10.1007/s11240-024-02688-7